Influence of caffeic acid phenethyl ester on bone healing in a rat model

Rat critical size cranial model and surgical procedure

Male Sprague–Dawley rats (n = 63; mean age 7 weeks; weight 280–490 g) were included in this study, which was conducted at the Health Institution of Research Centre, Dicle University, Diyarbakir, Turkey. Animals were individually housed in plastic cages in a controlled environment (21℃; 12-h light–dark cycle) with free access to drinking water and a diet of standard laboratory rat food pellets.

The rats were randomly divided into three groups: group A (control group), critical size cranial model with no treatment (n = 21); group B, critical size cranial model treated with CAPE (10 µmol/kg, daily intraperitoneal injection; n = 21); and group C, critical size cranial model treated with CAPE (10 µmol/kg, daily intraperitoneal injection) and β-tricalcium phosphate/hydroxyapatite bone graft substitute (n = 21).

Animals were anaesthetized with 5 mg/kg ketamine hydrochloride (Ketavett, Ratiopharm, Ulm, Germany) by intramuscular injection at the surgical site. A midline incision was made from the nasofrontal area to the external occipital protuberance along the midsagittal suture. The skin and underlying tissues including the temporal muscle were reflected bilaterally to expose the full extent of the calvaria. A calvarial through-and-through osteotomy 7.0 mm in diameter was trephined into the dorsal portion of the parietal bone on each side of the midsagittal suture using a dental handpiece and a trephine bur (Messeinger, Dusseldorf, Germany) under constant irrigation with sterile saline, and the trephined bone was removed from the surgical field.

Straumann® BoneCeramic (particle size 500–1000 μm; Institut Straumann AG, Basel, Switzerland), a medical grade, particulate form of biphasic calcium phosphate comprising 60% hydroxyapatite and 40% β-tricalcium phosphate with a wide spongiosa structure, a wide interconnected pore system (300–1500 µm) and a crystalline size of 10–60 μm, was moistened with sterile saline for 5 min before being placed into the defect to fill in the defect area.

Periosteum and skin were sutured in two layers using resorbable suture (Vicryl 3.0; Ethicon GmbH and Co. KG, Norderstedt, Germany). Upon completion of the surgical procedure, each animal received a single dose of 100 mg/kg ampicillin antibiotic by intramuscular injection.

Following surgery, seven rats per study group were killed at day 7, 15 and 30, using high dose ketamine. Bone regeneration, fibrotic healing and osteoblast activity were evaluated by histopathology.

All animal procedures were approved by the Animal Research Committee of Dicle University, Diyarbakır, Turkey and performed in compliance with the Medical Research Centre University guidelines for the care and handling of experimental animals.

Histopathological evaluation

Rat calvaria were fixed in 10% formaldehyde solution for 48 h and decalcified with 5% nitric acid. Calvarial specimens were washed with tap water, dehydrated with ascending concentrations of ethylalcohol, cleaned in xylene (Sigma-Aldrich, Steinheim, Germany) and infiltrated with paraffin. Starting from the centre of each specimen, serial sections (5 µm) were cut parallel to the midsagittal suture defect using a microtome, and stained with haematoxylin and eosin.

Tissue samples were assessed by a pathologist using a light microscope (Nicon Eclipse 80i, SEQ Enterprises, South Lakeland, FL, USA). The pathologist was blinded to the treatment group that each specimen came from. A 10-point scale ¹⁴ was used for evaluation of bone healing, where tissue was scored according to histopathological findings in the healing zone, as follows: 1, only fibrous tissue; 2, predominantly fibrous tissue; 3, equal amounts of fibrous and cartilage tissue; 4, predominantly cartilage tissue with little fibrous tissue; 5, only cartilage tissue; 6, predominantly cartilage tissue with little immature bone; 7, equal amounts of cartilage and immature bone tissue; 8, predominantly immature bone with little cartilage tissue; 9, healing with immature bone; 10, healing with mature bone.

Statistical analyses

Data were analysed using the SPSS® software package, version 15.0 (SPSS Inc., Chicago, IL, USA) for Windows®. Shapiro–Wilk test and graphical methods were used to test normality of the data. Kruskal–Wallis and Bonferroni-corrected Mann–Whitney U-test were used for between-group comparisons at each time point, and within-group comparisons between each time point. Spearman’s correlation coefficient analysis was performed to analyse correlation between the treatment groups across all three time points. For the Bonferroni-corrected Mann–Whitney U-test analysing differences between the three groups, a P-value < 0.0167 was considered statistically significant. In all other cases, a P-value < 0.05 was considered statistically significant.